Apparent-genetics, also known as pseudo-genetics, epigenetics, ex-genetics and metagenetics, is a biology discipline that investigates the reversible, heritable changes in gene function in case of the DNA sequences of a cell nucleus are not changed. It refers to the functional modification of the genome without changing the nucleotide sequence. Epigenetic phenomena include DNA methylation, RNA interference, tissue protein modification, and so on.
The post-transcription modification of histone mainly includes acetylation, methylation, phosphorylation, polyubiquitination and SUMO acylation of the histone, in which acetylation is one method that is studied most widely. Acetylation and deacetylation of histone play a key role in the process of structural modification of nuclear chromatin, which are regulated by the activities of histone acetyltransferase (HAT) and histone deacetylase (HDAC) (Saha, R. N. Pahan, K., Cell Death Differ 2006, 13 (4), 539-50).
Up to date, 18 human HDACs have been found and identified, and they are divided into four classes based on their similarity to yeast HDAC. The classes are type I (HDAC 1, 2, 3 and 8), type II (IIa: HDAC 4, 5, 7 and 9, IIb: HDAC 6 and 10), and type IV (HDAC 11), the activity of all these types depend on Zn2+. For type III HDACs (SirT 1-7), the enzyme activity depends on NAD+. (Karagiannis, T. C., El-Osta, A. Leukemia 2007, 21(1), 61-5.)
The histone deacetylase inhibitors (HDACi) involves in the regulation of the following important biological functions, including: 1) inducing apoptosis through exogenous or intrinsic apoptosis mechanisms; 2) blocking cell cycle; 3) inhibiting the neovascularization; 4) acetylation of tubulin and destruction of aggregate formation; 5) changing the tubulin structure to affect cell motility and differentiation; 6) regulating tumor immunity by the way of influencing the function of T cell receptors, the cytokine environment of immune effector cells, and directly up-regulating the other immune effector to identify the tumor cell protein, etc. (Zain J., Hematol Oncol Clin Northam, 2012, 26 (3): 671-704.) HDAC dysfunction may lead to imbalance of histone acetylation, so as to change the chromatin structure, and make the cell growth, differentiation, and apoptosis-related gene expression be inhibited, and finally lead to tumor formation. Currently, HDAC is an important target for the development of new antitumor drugs. In 2006, FDA approved SAHA (Vorinostat) as the first marketed HDACi for the treatment of cutaneous T-cell lymphoma (CTCL). In 2009, FK228 was marketed as a drug for treating CTCL and peripheral T-cell lymphoma (PTCL).
Recent studies have shown that HDACi may also be associated with a variety of autoimmune diseases. Early in 2003, Pahan et al. reported that HDAC inhibitor sodium phenylbutyrate can alleviate the central nervous system injury in the animal models of multiple sclerosis (MS) mice (experimental autoimmune encephalomyelitis, EAE), but did not explain the direct relationship between this result and the HDAC; two years later, Camelo et al. found that HDACi TSA can effectively inhibit the invasion of T cells to the mice central nervous system, he stressed that because of the inhibition of TSA against HDAC, the expression of neuroprotective protein such as IGF-2 and glutamate transporter EAAT2 and so on was increased, and thus a therapeutic effect was achieved; then there are many researchers who found the application of HDACi to MS, e.g., the studies of Ryu et al. showed that selective HDACi can increase the acetylation of the transcription factor Spl to protect the neuronal cells survival against oxidative stress (Giuseppe Faraco, et al., Molecular Medicine, 2011, 17 (5-6), 442-447). In view of the unknown mechanism of MS and the lack of sensitive diagnostic markers currently, HDACi may actively promote the treatment of MS. In addition, according to the report (Charles A Dinarello, et al., Molecular Medcine, 2011, 17(5-6), 333-352), HDACi is also associated with type 2 diabetes and its associated complications, neurodegenerative diseases (Huntington's disease, Alzheimer's disease) and so on, so HDAC is a target with a good research prospects.
The currently studied HDAC inhibitors mainly comprise three moieties: a chelating moiety with Zn2+ (ZBG), a hydrophobic linking moiety (Linker) and a surface recognition structural domain. According to the various zinc ion chelating groups, they can be divided into hydroximic acids, o-phenylenediamines, electron-deficient ketones, short-chain fatty acids and so on. According to the data from Thomson Reuters in December 2013, there are more than 100 HDACi being at different stages of drug research and development. The first listed SAHA is the hydroxypentanoic acid HDAC inhibitor which is used in the treatment of CTCL. With the further use, its shortcomings are exposed: the treatment effect of single drug is only average, it is not the first-line drug, the toxicity in high doses is obvious, and accompanied by the side effect of prolonged QT interval, bone marrow suppression, diarrhea and so on, and the treatment effect on solid tumor is not desired. This may be due to the fact that SAHA is a pan-inhibitor, and probably because it contains a strong zinc-ion chelating group, i.e., hydroximic acid group. Therefore, it is an important research direction in the art to develop newer and more efficient HDAC inhibitors.
The inventors of the present application filed a patent application (WO2012152208) in 2012, and reported a novel thiazole-based compound which can be used as HDAC inhibitors for the development of anti-tumor and multiple sclerosis drugs. In which, the compound CFH367-C showed good enzymatic inhibition activity, GI50 on HCT-116 cell was less than 1 μM, and the clinical symptoms of EAE mice were effectively relieved. However, due to the shortcomings of hydroxamic acid groups, it is desirable to develop a more active, less toxic HDAC inhibitor.
Based on the basic structure of the HDAC inhibitor, the inventors started from replacing the zinc ion chelating group (ZBG), firstly, the hydroxamic acid in the CFH367-C was replaced with the common o-phenylenediamine, but the obtained compounds has the enzyme level of IC50 decreased from 60 nM to 2-5 μM. After replacing the ZBG with a trifluoromethyl ketone and even no reported hydrazone compounds, the zinc ion chelating ability was reduced, however, it was unexpectedly found that these compounds has a higher enzyme inhibition activity (IC50=30 nM), a more inhibitory activity at the cellular level (IC50 up to 100 nM), and the treatment effect on clinical symptoms in EAE mice is significantly better than CFH367-C (the FIGURE), which shows a better development prospect.